The present invention relates, in particular, to human flavin monooxygenase 2 (hFMO2), as well as to another human enzyme of the FMO family, i.e. hFMOx, and to their nucleotide and polypeptide sequences. The present invention also relates to cloning and/or expression vectors which contain said nucleotide sequences and to cells which are transformed with these vectors, as well as to methods for preparing said polypeptides. The invention also encompasses methods for selecting compounds and for diagnosing predisposition to pathologies and/or deficiencies which are linked to the FMOs as well as to pharmaceutical compositions which comprise said compounds, which are intended for treating and/or preventing these pathologies.
The flavin monooxygenases (FMOs) (Lawton et al., 1994) form a family of microsomal enzymes which catalyze the NADPH-dependent oxidation of a large number of exogenous organic compounds (xenobiotics) which possess a nucleophilic heteroatom such as, in particular, the nitrogen, the sulfur, the phosphorus or the selenium atom (Ziegler D. M., 1988; Ziegler D. M., 1993), whether the xenobiotics are drugs, pesticides or other potentially toxic substances. Cysteamine is currently the only known endogenous substrate of the FMOs.
The FMOs represent a multigenic family. Expression of different forms of FMO depends both on the tissue and the species under consideration.
FMOs have been located in various types of tissue, in particular the liver, the lungs and the kidneys.
To date, five isoforms of FMO have been characterized in the reference species, which is the rabbit. Their homology is 50-60%. Four of these isoforms, i.e. FMO1, FMO3, FMO4 and FMO5, have been identified in humans (GeneBank sequences M64082, M83772, Z11737 and L37080, respectively). Among the mammalian species, the homology between orthologous FMOs is greater than 80%. It is reasonable to postulate that an FMO2, if not to say other isoforms, exist(s) in humans.
The FMOs are associated with the endoplasmic reticulum and are involved in detoxifying xenobiotic compounds, with monooxygenation enabling the xenobiotic to be transformed into a more polar substance, with this transformation being the preliminary step prior to its excretion. The FMOs may also be involved in the metabolic activation of various toxic and/or carcinogenic compounds which are present in the environment.
The mechanism of the FMO reaction has been described in detail (Poulsen, L. L. et al., 1995). In contrast to all the other known oxidases or monooxygenases, the FMOs possess the unique property of forming a stable, NADP(H)- and oxygen-dependent enzyme intermediate, i.e. 4xcex1-hydroperoxyflavin, in the absence of oxidizable substrate. Because the catalytic energy is already present in the FMO enzyme before contact with its potential substrate, the appropriateness of the substrate does not have to be as precise as in the case of other types of enzyme. This specific characteristic of FMO is responsible for the large variety of substrates which are accepted by the FMOs (including, for example, tertiary and secondary alkylamines and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides and thiols).
Many molecules which are active compounds of drugs are recognized as being substrates of the FMOs, either for an N oxidation or for an S oxidation (Gasser, 1996), with these molecules including, in particular, antidepressants, neuroleptics, anti-ulcer drugs, vasodilators and antihypertensives.
Although some FMO substrates are oxidized into less active derivatives, a large number of nucleophilic compounds can be metabolized into intermediates which may be more reactive and/or potentially toxic; rather than being excreted, such products may induce toxic responses by means of covalent binding to cell macromolecules, or by means of other mechanisms. For example, mercaptopyrimidines and thiocarbamides may be mainly activated by FMO activity (Hines et al., 1994). More precisely, it has been demonstrated that the nephrotoxicity which is associated with the glutathione conjugate of acrolein is linked to its metabolism mediated by renal FMO; the FMO forms an S-oxide which is then released, by an elimination reaction which is catalyzed in basic medium, in the form of cytotoxic acrolein (Park, S. B. et al., 1992). Thus, the FMOs can play an important role both in the first steps of chemical toxicity and in the detoxification of xenobiotic compounds.
As described above, a large number of drugs which are currently at the clinical trial stage, or else widely prescribed, contain nucleophilic functions of the nitrogen, sulfur, phosphorus or other type. However, the role of FMO in the oxidative metabolism of drugs and endogenous chemical compounds in humans is not well understood.
Cashman et al. (1996) have recently studied the contributions of the FMO enzymes in the physiological metabolism of cimetidine and S-nicotine in vivo. The greater part of their results confirms the fact that the FMO3 activity of the adult liver is responsible for the oxygenation of cimetidine and S-nicotine, with this oxygenation being stereospecific. The authors furthermore demonstrate that the stereochemistry of the main metabolites of cimetidine and S-nicotine in small experimental animals is different from that observed in humans and suggest that different FMO isoforms may predominate depending on the species, with this possibly having important consequences with regard to the choice of experimental animals for programmes for elaborating and developing drugs for humans.
FMO1 is known to be expressed in humans in the kidneys but not in the liver. FMO2 is expressed in the main in the lungs in all the mammalian species tested. In humans, FMO3 was isolated from the liver, where it predominates in adults. FMO3 is the main isoform involved in the sulfoxidation of methionine and in the stereospecific oxygenation of cimetidine and S-nicotine. FMC3 exhibits a greater specificity for its substrate than that exhibited by the FMO1 enzymes which are found in the livers of most animal species studied. FMO4 is a minor isoform whose function and substrate specificity are not well known. It is present in the human liver and is also expressed in the brain, where it could be involved in the oxidation of antidepressant drugs such as imipramine. FMO5 is expressed in the human liver to a lower extent than is FMC3. Its apparent lack of efficacy as an enzyme involved in the metabolism of drugs suggests that it could be involved in a physiological function.
The differing expression profiles of the FMO isoforms, depending on tissues and/or species, therefore probably constitute a significant factor contributing to the differences in FMO activity which are observed between tissues and/or between species. Thus, the variety of FMO forms could have a significant impact on the differences in the responses of tissues and/or species to exposure to a xenobiotic compound. This is because the differences which are observed between tissues and/or species in the response to xenobiotic compounds, and in the toxicity of these compounds, are linked, to a substantial extent, to variations in the activity and specificity involved in the metabolism of these substrates by the FMOs. Genetic factors and tissue specificity in the expression of the FMOs are important factors in these variations.
With regard to genetic factors, it has been reported, for example, that trimethylaminuria, which is a condition which is present in 1% of white British subjects and which is expressed in a strong odor of rotting fish in the expired air, the sweat or the urine, is linked to a deficiency of genetic origin in the functioning of an hepatic FMO.
For the reasons which have previously been mentioned, there therefore currently exists a considerable need to identify new isoforms of FMO, as well as the genetic polymorphisms which may be associated with them, which exhibit specificities with regard to their substrates and/or their tissue expression profile, which could be involved in the metabolism of xenobiotics, such as the metabolism of drugs or of exogenous substances which are present in the environment, such as, for example, pesticides, or else which could be involved in a physiological function. This is precisely the object of the present invention.